Heat-treated limulus amebocyte lysates

ABSTRACT

The application provides heat-treated  Limulus  amebocyte lysates useful for detecting β-glucans.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/220,785, filed Jun. 26, 2009, the complete contents of which areincorporated by reference herein.

BACKGROUND OF THE INVENTION

Limulus Amebocyte Lysate (LAL) derived from the blood cells of theAmerican horseshoe crabs react with endotoxin (Levin et al. (1964) Bull.Johns Hopkins Hosp. 115:265-274) or (1→3)-β-D-glucan (Kakinuma et al.(1981) Biochem. Biophys. Res. Commun. 101:434-439; Morita et al. (1981)FEBS Lett. 129:318-321), forming a gel. Endotoxin and β-glucan triggertwo distinct LAL pathways, activation of either of which leads togelation (Iwanaga et al. (1992) Thromb. Res. 68:1-32). Endotoxin is acell wall component of Gram-negative bacteria, and is pyrogenic,mitogenic, and potentially lethally toxic. Accordingly, accurate andreliable detection of endotoxin is important for confirming the safetyof parenteral drugs. LAL assays are the accepted standard for endotoxindetection (see, for example, the United States Pharmacopeia “BacterialEndotoxins Test”).

Because LAL reacts with either endotoxin or β-glucan, knowing which ofthe two activators is present in a sample is not necessarilystraightforward. β-glucan is commonly found in fungi, yeast, algae, andplants, and causes false positives in Bacterial Endotoxin Tests.β-glucan contamination has been reported in parenteral drugs, such asblood products (Ikemura et al. (1989) J. Clin. Microbiol.27(9):1965-1968), and medical devices, such as hemodialyzers (Peason etal. (1984) Artif. Organs 8:291-298). The detection of β-glucancontamination in parenteral drugs and medical devices is useful to avoidunexpected rejection of a product that should not be rejected (Cooper etal. (1997) J. Parenter. Sci. Technol. 51:2-6).

β-glucan detection can also be used on human blood samples to assist inthe diagnosis of deep mycosis (Obayashi et al. (1995) Lancet 345:17-20;Mori et al. (1997) Eur. J. Clin. Chem. Clin. Biochem. 35:553-560;Odabasi et al. (2004) Clin. Infect. Dis. 39:199-205; Ostrosky-Zeichneret al. (2005) Clin. Infect. Dis. 41:654-659). The FDA and the Japanesegovernment have each approved such assays as clinical diagnosticmethods.

The challenge has been to develop reliable, cost-effective assays forβ-glucan detection that can distinguish the presence of β-glucan fromthe presence of endotoxin. Several processes for increasing the β-glucanspecificity of LAL have been reported.

Fractionation methods (Obayashi et al. (1985) Clin. Chim. Acta149:55-65; Kitagawa et al. (1991) J. Chromatography 567:267-273; andU.S. Pat. No. 5,681,710) are based on removing the endotoxin sensitivefactor (“Factor C”) from LAL by column chromatography, or by separationsteps between solid absorbents and LAL. The column chromatography andseparation steps must be performed aseptically. Thus, these systems canrequire expensive instruments and significant efforts to preventcontamination of the system with endotoxin or β-glucan.

Another approach suppresses endotoxin activity in samples usingendotoxin-neutralizing peptides (U.S. Pat. Nos. 5,616,557; 5,622,833;and 5,750,500). Efforts to suppress endotoxin activity can be affectedby high amounts of endotoxin, and require the addition of anti-endotoxinsubstances that may be expensive and contaminated with endotoxin orβ-glucan.

U.S. Pat. No. 5,571,683 adds an endotoxin neutralizing peptide to aβ-glucan assay with LAL. This method requires the purification of anendotoxin neutralizing peptide from the blood of horseshoe crabs. Thepurification can be expensive, and avoiding contamination with endotoxinand β-glucan during the purification can be difficult.

Similarly, U.S. Pat. No. 5,266,461 adds an antibody against Factor C.This method requires preparation, synthesis and purification ofantibody, again adding expense and the risk of contamination withendotoxin or β-glucan during the purification process.

Thus, despite the efforts over the past two decades to develop β-glucanassays, there remains a need for a simple method for preparing LAL witha reduced sensitivity to endotoxin.

SUMMARY OF THE INVENTION

It has been reported that heating lysates from Asian horseshoe crabs(Tachypleus tridentatus) to temperatures above 40° C. quicklyinactivated the β-glucan-sensitive factor (“Factor G”) (Muta et al.(1995) J. Biol. Chem. 270:892-897). It has now been discovered that thesame is not true of lysates harvested from the American horseshoe crab,Limulus polyphemus. Rather, Limulus Factor G appears to be comparativelyheat-stable. Specifically, when LAL is heated to a temperature above 40°C., sensitivity of the LAL to endotoxin is lost more quickly thansensitivity of the LAL to β-glucan. Exploiting the differentialheat-sensitivity of the endotoxin and β-glucan pathways in LAL permitsthe preparation of lysates with reduced reactivity to endotoxin, whileretaining reactivity to (1→3)-β-D-glucan.

Accordingly, in one aspect, the invention relates to methods ofpreparing a Limulus amebocyte lysate (LAL) with increased specificityfor (1→3)-β-D-glucan. The methods include heating the LAL to atemperature above 40° C. to reduce reactivity to an endotoxin. Theinvention also relates to methods of preparing a Limulus amebocytelysate with reduced sensitivity to an endotoxin. The methods includeheating the LAL to reduce Factor C activity in the lysate whileretaining reactivity to (1→3)-β-D-glucan. The heating conditions can beselected to provide a lysate in which reactivity to endotoxin is onlymildly reduced, or in which endotoxin reactivity is substantiallyeliminated, affording a lysate that is (1→3)-β-D-glucan-specific.

Importantly, if excessive heating (such as 56° C. for 30 minutes) isavoided, reactivity of the LAL to (1→3)-β-D-glucan can be retained. Forexample, the resulting lysate may remain sensitive to concentrations ofcarboxymethylated pachyman (a (1→3)-β-D-glucan) of 100 ng/mL; 10 ng/mL;1 ng/mL; 100 pg/mL; 10 pg/mL; 1 pg/mL; or 0.1 pg/mL.

In some embodiments, the (1→3)-β-D-glucan reactivity of the LAL isreduced by a factor of not more than 1,000 (i.e. retaining at least 0.1%of the original reactivity), not more than 100 (retaining at least 1% ofthe original reactivity), not more than 50 (retaining at least 2% of theoriginal reactivity), not more than 10 (retaining at least 10% of theoriginal reactivity), not more than 5 (retaining at least 20% of theoriginal reactivity), or not more than 2 (retaining at least 50% of theoriginal reactivity).

The reduction in endotoxin reactivity should exceed reductions inreactivity to (1→3)-β-D-glucan. If a substantial reduction in endotoxinreactivity is desired, the reduction can be by a factor of at least 10(i.e. retaining no more than 10% of the original reactivity), at least50 (retaining no more than 2% of the original reactivity), at least 100(retaining no more than 1% of the original reactivity), at least 500(retaining no more than 0.2% of the original reactivity), at least 1,000(retaining no more than 0.1% of the original reactivity), at least 5,000(retaining no more than 0.02% of the original reactivity), at least10,000 (retaining no more than 0.01% of the original reactivity), atleast 100,000 (retaining no more than 0.001% of the originalreactivity), or at least 1,000,000 (retaining no more than 0.0001% ofthe original reactivity).

Typically the methods involve heating the LAL to between 40° C. and 80°C., although at higher temperatures (particularly at or above 56° C.),heating times should be corresponding brief to avoid also rendering theLAL insensitive to (1→3)-β-D-glucan. For example, at temperatures above56° C. the heating time is preferably less than one minute. In contrast,depending on the desired degree of reduction in endotoxin reactivity, ifthe temperature is no more than 45° C., heating times exceeding 40minutes may be used.

In certain circumstances, the LAL is heated for at least a minimum timeto achieve a desired reduction in endotoxin reactivity. For example, theheating time may be selected to exceed t_(A1) hours, wheret_(A1)=0.825*2.718^((56340/(T+273)))/(9.54*10⁷⁶), where T is thetemperature in ° C., in some cases achieving an approximately 1000-foldreduction in endotoxin reactivity. Alternatively, the heating time maybe selected to exceed t_(A2) hours, wheret_(A2)=1.65*2.718^((56340/(T+273)))/(9.54*10⁷⁶), in some cases achievingan approximately 1,000,000-fold reduction in endotoxin reactivity. Moregenerally, the LAL is optionally heated for at least t_(A) hours, wheret_(A)=K*0.275*2.718^((56340/(T+273)))/(9.54*10⁷⁶); K may be, forexample, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, or more, depending on the desired magnitude ofendotoxin reactivity inactivation.

In the methods described above, the LAL heating time optionally is nomore than t_(B1) hours, wheret_(B1)=0.353*2.718^((76510/(T+273)))/(2.20*10¹⁰³), where T is thetemperature in ° C., in some cases retaining at least approximately 10%of reactivity to (1→3)-β-D-glucan. If high levels of reactivity are notrequired for a particular application, longer heating times can betolerated. In various embodiments, the LAL heating time is optionally nomore than t_(B) hours, wheret_(B)=K*0.177*2.718^((76510/(T+273)))/(2.20*10¹⁰³), where K is 10.0,9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0,2.5, 2.0, 1.5, 1.0 or less, depending on the tolerance for inactivationof (1→3)-β-D-glucan reactivity.

After heating according to any of the methods described above, anyprecipitate is removed from the lysate, such as by centrifugation. Thelysate can also be prepared for use or storage, for example by admixingone or more additives (such as salts and/or buffer), or bylyophilization. Preferably, neither endotoxin neutralizing peptides noranti-Factor C antibodies are added, as these can add complexity and costto the process of preparing the heat-treated LAL.

In another aspect, the invention provides a heat-treated LAL comprising(1→3)-β-D-glucan-sensitive Limulus Factor G. The LAL has an endotoxinreactivity less than 0.1% of the endotoxin reactivity of an untreatedLimulus amebocyte lysate. The LAL may be lyophilized, and preferablyremains sensitive to 100 pg/mL, 10 pg/mL, 1 pg/mL, or even 0.1 pg/mL ofcarboxymethylated pachyman, while preferably being insensitive to 200EU/mL of Reference Standard Endotoxin, and/or to 0.01, 0.1, 1, or even10 mcg/mL of lipopolysaccharide (LPS).

To facilitate the detection of a (1→3)-β-D-glucan, the invention alsoprovides a composition including a heat-treated Limulus amebocyte lysateand a substrate which, upon activation of the LAL, yields a detectableproduct indicative of the activation of the lysate. The substrate may,for example, be chromogenic, presenting a change in absorbance at aparticular wavelength or spectrum of wavelengths upon activation of thelysate. Alternatively, the substrate may be fluorogenic, withfluorescent properties that change (increasing or decreasing or changingwavelength, for example) upon activation of the lysate.

The invention also provides methods for detecting a (1→3)-β-D-glucan ina sample. The methods include combining the sample with a substrate anda heat-treated Limulus amebocyte lysate according to any of the abovedescriptions. The methods can include detecting a change in an opticalproperty (such as absorbance, transmittance, fluorescence or turbidity)of the sample after the combination with the substrate and heat-treatedLAL.

The invention will be further understood in view of the drawing,specification, and claims which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical depiction of the relationship between onset timeand carboxymethylated pachyman concentration for an untreated LAL.

FIG. 2 is a graphical depiction of the relationship between onset timeand endotoxin concentration for an untreated LAL.

FIG. 3 is a graphical depiction of the relationship between onset timeand carboxymethylated curdlan concentration for an untreated LAL or LALheated to 47° C. for 30, 45, 60, or 75 minutes.

FIG. 4 is a graphical depiction of the relationship between onset timeand endotoxin concentration for an untreated LAL or LAL heated to 47° C.for 30, 45, 60, or 75 minutes.

FIG. 5 is a graphical depiction comparing standard curves ofcarboxymethylated curdlan with and without of 200 EU/mL of US ReferenceStandard Endotoxin.

FIG. 6 is a graphical depiction comparing standard curves ofcarboxymethylated curdlan with and without of 500 ng/mL of endotoxinderived from E. coli O55:B5.

FIGS. 7A-7D are schematic illustrations in perspective view (FIG. 7A),top view (FIG. 7B), side view (FIG. 7C), and end view (FIG. 7D), of anexemplary cartridge useful in performing β-glucan assays with theheat-treated LAL of the present invention.

FIG. 8 is an Arrhenius plot relating the rates of lysate inactivationobserved in Example 2 with absolute temperature measured in Kelvins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for preparing Limulus amebocytelysates with enhanced specificity for (1→3)-β-D-glucan and reducedsensitivity to endotoxin. This objective is achieved without the needfor additives such as endotoxin neutralizing peptides or antibodies, andwithout the need for exposure to chromatographic media. In this way,unnecessary expense and labor are avoided. Perhaps more importantly,unnecessary exposure of the LAL to potential sources of contamination isalso avoided. The present invention provides a simple, controlledheating step, which can be done on a crude lysate sealed, for example,in a vial or tube, creating little opportunity for contamination. TheLAL can subsequently be centrifuged to remove any precipitate andlyophilized for storage and later use in a β-glucan detection assay.

As used herein, the term, “(1→3)-β-D-glucan” is understood to mean anypolysaccharide or derivative thereof that is (i) capable of inducingformation of a coagulin clot in Limulus amebocyte lysate, and (ii)contains β-D glucosides, connected by a (1→3)-β-D glycosidic linkage. Itis contemplated that such a polysaccharide or derivative thereof, inaddition to containing a (1→3)-β-D glycosidic linkage, may also containglucoside moieties connected by a variety of other glycosidic linkages,for example, via a (1→4)-β-D glycosidic linkage and/or by a (1→6)-β-Dglycosidic linkage. It is contemplated that such (1→3)-β-D-glucans maybe isolated from a variety of sources including, without limitation,plants, bacteria, yeast, algae, and fungi, or alternatively may besynthesized using conventional sugar chemistries.

As used herein, Limulus amebocyte lysate, or LAL, can include a completeamebocyte lysate from Limulus polyphemus, or a fraction or componentthereof, naturally derived or recombinantly produced, containing Limuluspolyphemus Factor G and detectably reactive with a (1→3)-β-D-glucan.

Heat-Treatment of LAL

The LAL is generally heated while in a sealed container (for example, asealed tube or vial) to minimize the risk of contamination. The heat canbe applied in any controlled manner, such as immersion in atemperature-controlled water bath or heating block. For shorter heatingtimes, heating conditions can be selected to minimize the differentialbetween the temperature of the environment and the temperature of theLAL by, for example, using a sealed container with thinner and/or moreheat-conductive walls, reducing the volume of LAL, or using a containerwith a larger ratio of surface area/unit volume (such as a flattercontainer, a coiled container, etc.)

The LAL is heated to a temperature exceeding 40° C. As shown in Tables 1and 2 of Example 2 (in which increases in onset time are a measure ofdecreases in reactivity), at 40° C., endotoxin reactivity is slightlyreduced after 30 minutes, whereas β-glucan reactivity remainsessentially unaffected. In contrast, at 47° C., endotoxin reactivity islargely eliminated after only 20 minutes, whereas β-glucan reactivity isonly slightly reduced. Thus, across a range of temperatures endotoxinreactivity decreases more rapidly than β-glucan reactivity, permitting acontrolled reduction in endotoxin reactivity while preserving β-glucanreactivity. As the required sensitivity to β-glucan will vary (forexample, based on whether the detection is for clinical or industrialpurposes), the temperature and heating time can be selected to preservea desired level of β-glucan reactivity and/or to eliminate a desiredfraction of endotoxin reactivity. Although a range of temperatures canbe used, higher temperatures necessitate significantly shorter heatingtimes. For example, at temperatures above 56° C. the heating time ispreferably less than one minute (and, for increasingly elevatedtemperatures, substantially less than one minute).

Accordingly, temperatures greater than 40° C. but less than 56° C. canbe more convenient. On the other hand, as inactivation of endotoxinreactivity is comparatively slow 40° C.-45° C., often requiring inexcess of forty minutes to achieve substantial inactivation of endotoxinreactivity, temperatures greater than 45° C. (for example, 45° C.-55°C., 45° C.-52° C., or 45° C.-50° C.) can provide a more efficientprocess for preparing a (1→3)-β-D-glucan-specific LAL.

Typically the methods involve heating the LAL to between 40° C. and 80°C., although at higher temperatures (particularly at or above 56° C.),heating times should be corresponding brief to avoid also rendering theLAL insensitive to (1→3)-β-D-glucan. For example, at temperatures above56° C. the heating time is preferably less than one minute. In contrast,depending on the desired degree of reduction in endotoxin reactivity, ifthe temperature is no more than 45° C., heating times exceeding 40minutes may be used.

In certain embodiments, the LAL is heated to a maximum temperature above40° C. but no more than 45° C. (for example, 41° C.-45° C., 42° C.-45°C., 43° C.-45° C., 44° C.-45° C., or about 45° C.). In certainembodiments, the LAL is heated to a temperature above 45° C. (forexample, 46° C.-80° C., 46° C.-70° C., 46° C.-60° C., 46° C.-55° C., 46°C.-54° C., 46° C.-53° C., 46° C.-52° C., 46° C.-51° C., 46° C.-50° C.,46° C.-49° C., 46° C.-48° C., 46° C.-47° C., or about 46° C.). Heatingto temperatures less than 55° C. (for example, 41° C.-55° C., 41° C.-54°C., 41° C.-53° C., 41° C.-52° C., 41° C.-51° C., 41° C.-50° C., 41°C.-49° C., 41° C.-48° C., 41° C.-47° C., 41° C.-46° C.) or less than 53°C. (for example, 42° C.-53° C., 43° C.-53° C., 44° C.-53° C., 45° C.-53°C., 46° C.-53° C., 47° C.-53° C., 48° C.-53° C., 49° C.-53° C., 50°C.-53° C., 51° C.-53° C., or 52° C.-53° C.) provides the advantage of aslower degradation of (1→3)-β-D-glucan reactivity.

The heating time of the LAL can be varied depending on the temperatureand the desired level of endotoxin and (1→3)-β-D-glucan reactivities.For example, the heating time can be less than one minute; 1-5 minutes;1-10 minutes; 1-20 minutes; 1-30 minutes, 1-40 minutes; 1-50 minutes;1-60 minutes; 1-80 minutes; 1-100 minutes; 1-120 minutes; 1-240 minutes;at least five minutes; 5-10 minutes; 5-20 minutes; 5-30 minutes; 5-40minutes; 5-50 minutes; 5-60 minutes; 5-80 minutes; 5-100 minutes; 5-120minutes; 5-240 minutes; at least twenty minutes, 20-30 minutes; 20-40minutes; 20-50 minutes; 20-60 minutes; 20-80 minutes; 20-100 minutes;20-120 minutes; 20-240 minutes; more than forty minutes; 40-50 minutes;40-60 minutes; 40-80 minutes; 40-100 minutes; 40-120 minutes; 40-240minutes; two hours or less; or, at the lower temperatures, as long asseveral days.

In certain embodiments the lysate is tested to 40° C. for 750-13500minutes, or to 41° C. for 425-6200 minutes, or to 42° C. for 240-2900minutes, or to 43° C. for 137-1350 minutes, or to 44° C. for 78-620minutes, or to 45° C. for 45-290 minutes, or to 46° C. for 26-135minutes, or to 47° C. for 15-64 minutes, or to 48° C. for 8.5-31minutes, or to 49° C. for 5-15 minutes, or to 50° C. for 2.5-7 minutes,or to 51° C. for 1.5-3.5 minutes, or to 52° C. for 1-1.6 minutes, or to53° C. for 0.6-0.8 minutes.

As seen in the following Tables, the rate of inactivation of endotoxinreactivity varies dramatically based on the incubation temperature.Indeed, both the rate of inactivation of endotoxin reactivity and therate of inactivation of β-glucan reactivity increase exponentially withincreasing temperature. An analysis of the relationships among heatingtime, temperature, and rates of inactivation of endotoxin reactivity andβ-glucan reactivity is provided in Example 8. As discussed in Example 8,a series of experiments measured onset times for activation ofheat-treated LAL. The experiments varied heating time, temperature, andconcentration of endotoxin or β-glucan. The observed result for thereagents used was that the heating time required to achieve a 1,000-foldreduction in endotoxin reactivity was t_(A1) hours, wheret_(A1)=0.825*2.718^((56340/(T+273)))/(9.54*10⁷⁶), where T is thetemperature in ° C. Achieving a 1,000,000-fold reduction would requiretwice as long: t_(A2) hours, wheret_(A2)=1.65*2.718^((56340/(T+273)))/(9.54*10⁷⁶). The desired, requiredor permitted retention of endotoxin reactivity may vary from oneapplication to another (as it varies, for example, between industrialand diagnostic applications). Thus, more generally, a target heatingtime may be greater than or equal to t_(A) hours, wheret_(A)=K*0.275*2.718^((56340/(T+273)))/(9.54*10⁷⁶); K may be, forexample, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, or more, depending on the desired magnitude ofendotoxin reactivity inactivation.

Similarly, the rate of reduction of β-glucan reactivity increasesexponentially with increasing temperature, although with moderateheating temperatures the rate of loss for β-glucan reactivity can besubstantially smaller than the rate of loss for endotoxin reactivity.Thus, the observed result in Example 8 was that the heating time thatleads to a 10-fold loss of β-glucan reactivity was t_(B1) hours, wheret_(B1)=0.353*2.718^((76510/(T+273)))/(2.20*10¹⁰³), where T is thetemperature in ° C. Again, because in various applications the tolerancefor loss of β-glucan reactivity reactivity may vary, a target heatingtime may be less than or equal to t_(B) hours, wheret_(B)=K*0.177*2.718^((76510/(T+273)))/(2.20*10¹⁰³), where K is 10.0,9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0,2.5, 2.0, 1.5, 1.0 or less.

Following heat treatment, any precipitate can be removed from the lysateby centrifugation (at 2500×G for 20 minutes, for example), filtration,or other separation step.

The heat-treated LAL can also be dried onto a solid surface (such as avial, a cartridge or a 12-well or 96-well plate), such as bylyophilization. Prior to drying, one or more additives are optionallyadmixed with the heat-treated LAL. For example, a resolubilizing and/oran anti-flaking agent can be included. The resolubilizing agent is anagent that, either alone or in combination with another resolubilizingagent, facilitates the resolubilization of one or more components of theLAL once the LAL is exposed to a fluid sample. The resolubilizing agentpreferably also stabilizes the lysate in its dried form. Theresolubilizing agent provided in the mixture facilitates the stabilityof the reagents and their dissolution during the assay. Resolubilizingagents include, for example, one or more sugars, salts, or combinationsthereof. Preferred sugar resolubilizing agents include, for example,mannitol, mannose, sorbitol, trehalose, maltose, dextrose, sucrose, andother monosaccharides or disaccharides. The anti-flaking agent includedin the mixture further stabilizes the reagents and reduces flaking ofthe dried lysate. The anti-flaking agent preferably also stabilizes thelysate in its dried form. Preferred anti-flaking agents include, forexample, one or more polymers, for example, polyethylene glycol,polyvinyl pyrolidone, polyvinyl alcohol, mannitol, dextran, andproteins, for example, serum albumin. An anti-frothing agent such aspolyvinyl alcohol or polypropylene glycol can also be included. Saltsand/or buffers, such as sodium chloride, magnesium sulfate, and HEPESbuffer can also be included, as exemplified in Example 1. Other kinds ofbuffers, such as TRIS-HCl buffer, TES, MOPS, PIPES, BES, MOPSO, DIPSO,MOBS, TAPSO, HEPPSO, POPSO, TEA, EPPS, Tricine, and phosphate can beused, as can other buffers with buffering capacity between pH 7 and pH8, as this is a preferred range of pH for the reaction. The target pH ofthe composition, after admixture with a sample, is preferably between7.3 and 8.0.

The mixture can be dried onto a surface of the conduit in an environmenthaving a temperature of about 4° C. to about 40° C., more preferably,from about 10° C. to about 35° C., more preferably, from about 15° C. toabout 30° C. and a relative humidity of about 0% to about 30%, morepreferably, from about 2% to about 20%, more preferably, from about 4%to about 10%. Preferably, the temperature is about 25° C. and therelative humidity is about 5%. Drying preferably occurs for about 10minutes to about 8 hours, more preferably for about 1 hour in atemperature regulated drying chamber.

In another embodiment, the mixture is dried onto the surface of theconduit by lyophilization or freeze-drying, for example, at temperaturesbelow 0° C., for example, from about −75° C. to about −10° C., morepreferably from about −30° C. to about −20° C.

β-Glucan Assays

Heat-treated LAL can be used to detect β-glucan using any of a varietyof endpoint or kinetic assays. Exemplary endpoint assays include anendpoint chromogenic assay or an endpoint turbidimetric assay. Exemplarykinetic assays include a kinetic turbidimetric assay, a one-step kineticassay or a multi-step kinetic assay. Each of the assays is discussed inmore detail below. Furthermore, it is understood that the assays may bemodified to be performed in a particular assay format, for example, in acartridge or in the well of a plate, for example, a 96 well plate.

(1) Kinetic Assays

Exemplary kinetic assays include multi-step kinetic assays, single-stepkinetic assays, and kinetic turbidimetric assays.

(i) Multi-Step Kinetic Assay

A multi-step kinetic assay (for example, as described in U.S. Pat. No.7,329,538) is initiated by combining the sample to be tested with avolume of heat-treated LAL to produce a sample-LAL mixture. The mixturethen is incubated for a predetermined period of time. The mixture thenis contacted with a substrate, for example, a chromogenic or fluorogenicsubstrate, to produce a sample-LAL-substrate mixture. Thereafter, thetime in which a preselected change in an optical property (for example,a specific change in an absorbance value or a specific change in atransmission value) is measured.

The assay can be calibrated by measuring the time in which a preselectedchange in an optical property occurs when a certain amount ofcarboxymethylated pachyman or other β-glucan is introduced into theassay. By comparing the result generated by a test sample against theresults generated by one or more known amounts of β-glucan, it ispossible to detect the presence or amount of β-glucan in a test sample.

It is understood that a multi-step kinetic assay can be run in acartridge format. The cartridge preferably is used with an opticaldetector, for example, a hand-held optical detector as shown anddescribed in U.S. Pat. No. Des. 390,661.

By way of example and as illustrated in FIGS. 7A-7D, cartridge 1 has asubstantially planar housing fabricated, for example, from a moldablebiocompatible material. The housing may be fabricated from any material,however, transparent and/or translucent glass or polymers are preferred.Preferred polymers include, for example, polystyrene, polycarbonate,acrylic, polyester, optical grade polymers, or any plastic such that theoptical cell is substantially transparent. The housing contains at leastone fluid inlet port 4, at least one optical cell 6, and at least oneconduit 8 having a fluid contacting surface for providing fluid flowcommunication between the fluid inlet port 4 and optical cell 6. Theonly requirements for the optical cell 6 are that it defines a voidcapable of containing a sample to be tested and that a portion of theoptical cell 6 is transparent to light. Cartridge 1 may also have atleast one pump port 12 in fluid flow communication with fluid inlet port4 and optical cell 6 for attaching the cartridge 1 to a pump. The pumpmay then impart a negative pressure via pump port 12 to pull the samplefrom fluid inlet port 4 to optical cell 6. A heat-treated LAL isdisposed on a first region 14 of the fluid contacting surface of conduit8, so that when a sample is applied to fluid inlet port 4, the sampletraverses region 14 and solubilizes or reconstitutes the LAL into thesample as it moves toward optical cell 6.

A second region 16 of the fluid contacting surface of conduit 8 isspaced apart from and downstream of first region 14. In thisconfiguration, LAL is disposed at first region 14 and a chromogenic orfluorogenic substrate is disposed at second region 16, so that after thesample is contacted with the LAL in region 14, the sample-lysate mixturetraverses conduit 8 and contacts the substrate in region 16. Thesample-lysate-substrate mixture then traverses conduit 8 to optical cell6.

The cartridges can be designed and used according to the type and/ornumber of tests required. For example, a single sample may be tested,for example, in duplicate or triplicate, for example, for researchlaboratory use or for medical device and biopharmaceutical testing.Alternatively, two or more different samples may be tested individually.The cartridge preferably is a single-use, disposable cartridge that isdiscarded after one use. The cartridges typically use approximately20-100 fold less hemocyte lysate per sample than is used in theconventional endpoint chromogenic or kinetic chromogenic assaysperformed in multi-well plates, and thus provides a less costly andenvironmentally-friendlier test.

With reference to FIG. 7A, in order to perform a multi-step kineticassay in a exemplary cartridge 1, a sample is first moved, for example,by pump action, to a first region 14 containing the heat-treated LAL,where it is mixed and incubated for a predetermined period of time. Thesample-LAL mixture then is moved, for example, by pump action, to thesecond region 16 containing the substrate, for example, a chromogenic orfluorogenic substrate, where it is solubilized. The sample-substratemixture then is moved to optical cell 6, for a measurement of an opticalproperty. The time intervals required for mixing and incubating stepsare preprogrammed for optimal specificity and sensitivity to theβ-glucan concentration range of interest.

Although the multi-step assay may be performed in a cartridge of thetype discussed above, it may also be employed in a variety of otherformats, for example, within the well of a microtiter plate. In thistype of assay, a sample of interest is combined with a heat-treated LALand incubated for a predetermined period of time. Then, after thepredetermined period of time, a chromogenic or fluorogenic substrate isadded to the well. After mixing, the time in which a preselected changein an optical property occurs is measured. The result can then becompared against one or more standard values to measure the presence oramount of β-glucan in the sample.

In the well-type format, the samples and reagents are added to each ofthe wells, preferably using an automated system, such as a robot, andthe plate processed by a microplate reader, which can be programmed tosequentially read the absorbance of each well in a repetitive fashion.

(ii) Single-Step Kinetic Assay

A single-step kinetic assay, for example, a single step-chromogenicassay, is described in U.S. Pat. No. 5,310,657. Briefly, a kineticchromogenic assay includes the steps of (i) simultaneously solubilizinga heat-treated LAL with a sample to be analyzed and a substrate, forexample, a chromogenic substrate, (ii) incubating the resulting mixtureat a temperature of about 0° to about 40° C., preferably about 25° toabout 40° C., over a predetermined time range and (iii) measuring a timerequired for a calorimetric change to reach a pre-selected value orchange of the calorimetric readout, using a conventionalspectrophotometer.

This type of assay, like the multi-step kinetic assay, can be performedin a cartridge or a well-type format. A cartridge similar to thatdescribed above for the multi-step kinetic assay can be modified for usein single-step kinetic assay. With reference to FIG. 7A, chromogenic orfluorogenic substrate is applied, for example, to the surface of conduit8 at first region 14 together with the heat-treated LAL. To perform akinetic assay in cartridge 1 and in reference to FIG. 7A, a sample ismoved, for example, by pump action, to a first region 14 of the conduit8 containing both the LAL and substrate, where they are solubilized, forexample, by cycling between forward and reverse pump action. Thesample-LAL-substrate mixture then is moved to optical cell 6 formeasurement of an optical property, for example, the absorbance ortransmittance properties of the sample by an optical detector. Thedetector may determine how long it takes for each optical property toexhibit, for example, a 5% drop in optical transmittance. Results frommultiple assays, for example, two assays, can be averaged.

The assay can be calibrated by measuring the time in which a preselectedchange in an optical property occurs when a certain amount of β-glucanis introduced into the assay. By comparing the result generated by atest sample against one or more results with known amounts of β-glucan,it is possible to measure the presence or amount of β-glucan in the testsample.

This type of assay format may be employed in a variety of other formats,for example, within the well of a microtiter plate. In this type ofassay, a sample of interest is mixed with a heat-treated LAL and achromogenic or fluorogenic substrate. After mixing, the time in which apreselected change in an optical property occurs is measured. The resultcan then be compared against standard values to measure the presence oramount of β-glucan in the sample of interest.

(iii) Kinetic Turbidimetric Assay

A kinetic turbidimetric β-glucan assay can include the steps of (i)solubilizing a heat-treated LAL with a sample to be analyzed, (ii)incubating the resulting mixture at a temperature of about 0° to about40° C., preferably about 25° to about 40° C., over a predetermined timerange, and (iii) measuring a time required for either a turbidity changecaused by coagulation to reach a pre-selected value or a ratio in changeof the turbidity, using a conventional coagulometer, nepherometer, orspectrophotometer.

This type of assay, like the previous assays, can be performed in acartridge or a well-type format. A cartridge similar to that describedabove for the multi-step or single-step kinetic assays can be modifiedfor use in kinetic turbidimetric assays. With reference to FIG. 7A, nochromogenic or fluorogenic substrate needs to be applied to either firstregion 14 or second region 16.

Referring to FIG. 7A, in order to perform a kinetic turbidimetric assayin a cartridge 1, a sample is, for example, moved to a first region 14of the conduit 8 containing the heat-treated LAL, where it issolubilized, for example, by cycling between forward and reverse pumpaction. The sample-lysate mixture then is moved to optical cell 6 formeasurement of an optical property, for example, turbidity, bymeasuring, for example, the absorbance or transmittance properties ofthe sample-lysate mixture using an optical detector. The detector maydetermine how long it takes for each optical property to exhibit, forexample, a 5% drop in optical transmittance. Results from multipleassays, for example, two assays can be averaged.

The assay can be calibrated by measuring the time in which a preselectedchange in an optical property, for example, turbidity, occurs when acertain amount of β-glucan is introduced into the assay. By comparingthe result generated by a test sample against one or more results withknown amounts of β-glucan, it is possible to measure the presence oramount of β-glucan in the test sample.

This type of assay format may be employed in a variety of other formats,for example, within the well of a microtiter plate. In this type ofassay, a sample of interest is mixed with a heat-treated LAL. Aftermixing, the time in which a preselected change in an optical property,for example, turbidity, occurs is measured. The result can then becompared against standard values to measure the presence or amount ofβ-glucan in the sample of interest.

(2) Endpoint Assays

Exemplary endpoint assays include endpoint chromogenic or fluorogenicand endpoint turbidimetric assays.

(i) Endpoint Chromogenic or Fluorogenic Assay

Endpoint chromogenic or fluorogenic β-glucan assays can include thesteps of (i) solubilizing a heat-treated LAL with a sample to beanalyzed, (ii) incubating the resulting mixture at a temperature ofabout 0° to about 40° C., preferably about 25° to about 40° C., for apredetermined time, (iii) contacting substrate, for example, achromogenic or fluorogenic substrate, with the incubated sample-LALmixture, (iv) optionally adding a reaction inhibitor, for example,acetic acid, and (v) measuring, for example by calorimetric change, asubstance produced from the substrate by enzymatic activity.

This type of assay can be performed in a cartridge or in a well-typeformat. When an endpoint chromogenic or fluorogenic assay is performedin a cartridge 1 (see, FIG. 7A), a sample is moved, for example, to afirst region 14 of the conduit 8 containing the heat-treated LAL, whereit is solubilized, for example, by cycling between forward and reversepump action. Following a predetermined incubation period, the sample-LALmixture then is moved, for example, by pump action to a second region 16of the conduit 8 containing the chromogenic or fluorogenic substrate,where it is solubilized, for example, by cycling between forward andreverse pump action. The sample-LAL-substrate mixture optionally then ismoved to a third region containing a reaction inhibitor. Afterwards, thesample-LAL-substrate mixture is moved to optical cell 6 for measurementof an optical property, for example, the absorbance or transmittanceproperties of the sample by an optical detector. It is contemplated,however, that when performing an end-point chromogenic or fluorogenicassay in a cartridge it is not necessary to stop the reaction using areaction inhibitor. Under this type of assay, the final optical readings(endpoint readings) are recorded at a predetermined time.

The assay can be calibrated by measuring an optical property, forexample, absorbance or transmittance, when a certain amount of β-glucanis introduced into the assay. By comparing the result generated by atest sample against one or more results with known amounts of β-glucan,it is possible to measure the presence or amount of β-glucan in the testsample.

As discussed, this type of assay format may be employed in a variety ofother formats, for example, within the well of a microtiter plate. Inthis type of assay, a sample of interest is mixed with a heat-treatedLAL and incubated for a preselected period of time. Then, a chromogenicor fluorogenic substrate is added to the mixture and the sampleincubated for another period of time. Then a reaction inhibitor, forexample, acetic acid, is added to the sample, and an optical property ofthe sample, for example, absorbance or transmittance, is measured. Theresult can then be compared against standard values to measure thepresence or amount of β-glucan in the sample of interest.

(ii) Endpoint Turbidimetric Assay

End point turbidimetric β-glucan assays can include the steps of (i)solubilizing a heat-treated LAL with a sample to be analyzed, (ii)incubating the resulting mixture at a temperature of about 0° to about40° C., preferably about 25° to about 40° C., for a predetermined time,(iii) optionally adding a reaction inhibitor, for example, acetic acid,and (iv) measuring the increase in turbidity as a result of coagulation,if any, using a conventional coagulometer, nepherometer, orspectrophotometer.

Endpoint turbidimetric assays can be performed in a cartridge-typeformat. With reference to FIG. 7A, a sample is applied to cartridge 1and is moved, for example, to a first region 14 of the conduit 8containing the hemocyte lysate, where it is solubilized, for example, bycycling between forward and reverse pump action. The sample-lysatemixture then is moved to optical cell 6 for measurement of an opticalproperty, for example, turbidity, using an optical detector. Resultsfrom multiple assays, for example, two assays can be averaged.

The assay can be calibrated, for example, by measuring the turbidity ata preselected time when a certain amount of β-glucan is introduced intothe assay. By comparing the result generated by a test sample againstone or more results with known amounts of β-glucan, it is possible tomeasure the presence or amount of β-glucan in the test sample.

This type of assay format may also be run in other formats, for example,within the well of a microtiter plate. In this type of assay, a sampleof interest is mixed with a heat-treated LAL and incubated for apreselected period of time. The reaction can then be stopped by theaddition of an inhibitor. An optical property, for example, turbidity,of the sample then is measured at a preselected time point. The resultcan then be compared against standard values to measure the presence oramount of β-glucan in the sample of interest.

Endotoxin Detection

Heat-treatment of a lysate can also be performed to reduce thedifferential sensitivity of the lysate to endotoxin and β-glucan. Inthis way, the relative reactivity of the lysate to endotoxin and toβ-glucan can be titrated to provide a dual-specificity detectionreagent.

Assays for endotoxin can be performed using any of the assay formatsdescribed above in the context of β-glucan assays, except that thepositive control would be an endotoxin source, such as alipopolysaccharide preparation. Such an assay can be used to confirm thespecificity of a β-glucan-specific heat-treated lysate, or to detectendotoxin in a test sample using a lysate retaining sufficientsensitivity to endotoxin.

Specimen Collection and Preparation Considerations

In general, materials used to harvest, store, or otherwise contact asample to be tested, as well as test reagents, should be free ofmicrobial contamination, for example, should be pyrogen-free. Materialsmay be rendered pyrogen-free by, for example, heating at 250° C. for 30minutes. Appropriate precautions should be taken to protectdepyrogenated materials from subsequent environmental contamination.

The heat-treated LAL may be used to measure the presence or amount ofβ-glucan in a sample of interest, for example, in a fluid, for example,a fluid to be administered locally or systemically, for example,parenterally to a mammal, or a body fluid to be tested for infection,including, for example, blood, lymph, urine, serum, plasma, ascitesfluid, lung aspirants, and the like. In addition, the assays may be usedto detect β-glucan present on a surface. For example, the surface ofinterest is swabbed and the swab then is introduced into or dissolved inliquid. The liquid can then be assayed as described herein.

EXAMPLE 1 Reactivity of Crude LAL with Endotoxin and β-Glucan

Crude LAL was prepared by harvesting hemolymph from American horseshoecrabs (Limulus polyphemus). The resulting hemolymph was centrifuged at150 G for 15 minutes to collect amebocytes. The amebocytes were rinsedwith 3% sodium chloride and recentrifuged at 150 G for 15 minutes. Aftersecond rinsing with 3% sodium chloride and harvesting steps by thecentrifugation at 150 G for 15 minutes, the resulting amebocytes werelysed by osmotic shock with addition of water for injection, andresulting crude LAL stored at 2-8° C. until further use.

LAL for the measurement was prepared by adding 0.34 M sodium chloride,0.04 M magnesium sulfate, 0.35% (w/v) dextran, and 0.04 M HEPES buffer(pH 8.0) to 20% (v/v) crude LAL. Carboxymethylated pachyman(CM-Pachyman, Megazyme Lot 90501) as a β-glucan and Lipopolysaccharide(endotoxin) derived from E. coli O55:B5 (List Biological Lab, Inc., lot20315A5) were dissolved and diluted with water for irrigation (WFI,Baxter) to obtain a 10-fold dilution series. CM-Pachyman dilutions from0.001 to 1 ng/mL and endotoxin dilutions from 1 to 1000 ng/mL weremeasured.

After 0.05 mL of each of the samples were distributed in the wells of amicroplate (Charles River), 0.05 mL of LAL was added to each well.Thereafter, 0.01 mL of 5 mM of a chromogenic substrate(Ac-Ile-Glu-Gly-Arg-pNA•HCl) was added to each well. The microplate wasset on a microplate reader (ELx 808, BIO-TEK INSTRUMENT, Inc.), and themeasurement was started. Measurements were performed at 37° C.: theabsorbance of each well at 405 nm was monitored and the onset times weredetermined.

FIGS. 1 and 2 show the dose-response curves of CM-Pachyman (aβ-D-glucan) and endotoxin, respectively. Increased activation of LAL isreflected in a shorter “onset time” in kinetic LAL methods. As expected,the concentration of either endotoxin or carboxymethylated pachymanshowed good correlations with onset time.

EXAMPLE 2 Effects of Temperature on the Inactivation of the Endotoxinand β-Glucan Cascades

Each 1 mL of the crude LAL prepared as described in Example 1 wasdistributed in depyrogenated 13 mm glass tubes. The glass tubes wereincubated on an aluminum block heater at a temperature between 40° C.and 50° C. A glass tube was sampled at the scheduled incubation time,and was stored in a refrigerator. The glass tubes were centrifuged at170 G for 20 minutes with a refrigerated centrifuge. The supernatant inthe glass tubes was used as heat-treated crude LAL.

LAL for use in the assay was prepared as described in Example 1 by usingthe sampled crude LAL. A solution of 50 ng/mL endotoxin derived from E.coli O55:B5, and a solution of 100 ng/mL carboxymethlated curdlan(CM-curdlan, Wako Chemicals) (a β-D-glucan) were measured with LAL.Onset times were measured as described in Example 1. The results aresummarized in Tables 1 and 2.

TABLE 1 Effect of Temperature on the Reactivity of LAL with Endotoxin.Incubation time Onset time (sec) (min) 40° C. 43° C. 45° C. 47° C. 50°C. 0 245 245 373 167 245 5 not done not done not done not done 2751 10339 not done 680 812 >3600 20 345 423 905 >3600 >3600 30 350 not done1323  >3600 >3600

TABLE 2 Effect of Temperature on the Reactivity of LAL with β-glucan.Incubation time Onset time (sec) (min) 40° C. 43° C. 45° C. 47° C. 50°C. 0 343 343 367 409 343 5 not done not done not done not done 439 10296 not done 310 326 1367 20 282 300 319 406 >3600 30 276 not done 329502 >3600

Table 1 shows the effect of temperature on reactivity of the crude LALwith endotoxin. Higher temperature showed a faster decrease inreactivity of the crude LAL with endotoxin. Table 2 shows the effect oftemperature on reactivity of the crude LAL with β-glucan. The β-glucanreactivity of the crude LAL was relatively more stable with increasingtemperature than the endotoxin reactivity was. These data indicate thatan LAL reagent for measuring β-glucan without significant reactivitywith endotoxin can be prepared by using the difference in the rates ofinactivation of endotoxin reactivity and β-glucan reactivity.

EXAMPLE 3 Effect of Heating on Dose-Response Curves of Endotoxin andβ-Glucan

Each 5 mL of the crude LAL prepared as described in Example 1 wasdistributed in depyrogenated 10-mL glass vials. The glass vials wereincubated on an aluminum block heater at 47° C. A glass vial was sampledat the scheduled incubation time, and was stored in a refrigerator. Thecrude LAL in the vials was transferred to polypropylene centrifugetubes, and the centrifuge tubes were centrifuged at 2500 G for 20minutes with a refrigerated centrifuge. Supernatant in the centrifugetubes was used as heat-treated crude LAL.

LAL for use in the assay was prepared as described in Example 1 by usingthe sampled crude LAL. A dilution series of endotoxin derived from E.coli O55:B5 between 10 ng/mL and 10,000 ng/mL, and a dilution series ofCM-Pachyman between 1 pg/mL and 1000 pg/mL were measured with LAL. Theprocedures of the measurement were the same as described in Example 1.The results are summarized in FIGS. 3 and 4.

FIGS. 3 and 4 show the effect of heating on dose-response curves ofendotoxin and β-glucan (CM-Pachyman), respectively. There was noconsiderable difference in the dose-response curves for differentincubation times (30 minutes-75 minutes). On the other hand, thedose-response of endotoxin was significantly decreased by heating at 47°C.

EXAMPLE 4 Effect of Reference Standard Endotoxin on the Measurement ofβ-Glucan with Heat-Treated LAL

Each 3 mL of the crude LAL prepared as described in Example 1 wasdistributed in 10 depyrogenated 13 mm glass tubes. The glass tubes wereincubated on an aluminum block heater at 47° C. for 15 minutes. Theheated crude LAL was collected in polypropylene centrifuge tubes, andthe centrifuge tubes were centrifuged at 170 G for 20 minutes with arefrigerated centrifuge. Supernatant in the centrifuge tubes was used asheat treated crude LAL.

LAL for use in the assay was prepared as described in Example 1 by usingthe heated crude LAL. Dilution series of CM-Curdlan between 1 ng/mL and100 ng/mL were prepared with and without 200 EU/mL US Reference StandardEndotoxin (RSE). The dilutions were measured with LAL. The procedures ofthe measurement were same as described in Example 1. The results aresummarized in FIG. 5.

FIG. 5 shows the effect of RSE on the β-glucan standard curve. Thestandard curve was not affected by the addition of 200 EU/mL RSE.Considering that commonly measured endotoxin levels are less than 50EU/mL, normal endotoxin contamination does not interfere with theability of the heat-treated LAL to accurately measure β-glucan levels.

Example 5 Effect of Endotoxin Derived From E. coli O55:B5 on theMeasurement of β-Glucan with Heat-Treated LAL

Each 3 mL of the crude LAL prepared as described in Example 1 wasdistributed in 10 depyrogenated 13 mm glass tubes. The glass tubes wereincubated on an aluminum block heater at 47° C. for 20 minutes. Theheated crude LAL was collected in polypropylene centrifuge tubes, andthe centrifuge tubes were centrifuged at 170 G for 20 minutes with arefrigerated centrifuge. Supernatant in the centrifuge tubes was used asheat treated crude LAL.

LAL for use in the assay was prepared as described in Example 1 by usingthe heated crude LAL. Dilution series of CM-Curdlan between 1 ng/mL and100 ng/mL were prepared with and without 500 ng/mL endotoxin derivedfrom E. coli O55:B5. The dilutions were measured with LAL. Theprocedures of the measurement were same as described in Example 1. Theresults are summarized in FIG. 6.

FIG. 6 shows the effect of RSE on the standard curve of β-glucan. Thestandard curve was not affected by the addition of 100 ng/mL endotoxinderived from E. coli O55:B5. Considering that 500 ng/mL endotoxinderived from E. coli O55:B5 approximates 5000 EU/mL, normal levels ofendotoxin contamination would not interfere with the ability of theheat-treated LAL to accurately measure β-glucan levels.

Example 6 Kinetic Turbidimetric Assay with Heat-Treated LAL

Each 5 mL of the crude LAL prepared as described in Example 1 wasdistributed in depyrogenated 10-mL glass vials. The glass vials wereincubated on an aluminum block heater at 47° C. for 60 minutes. Thecrude LAL in the vials was transferred to polypropylene centrifugetubes, and the centrifuge tubes were centrifuged at 2500 G. for 20 min.with a refrigerated centrifuge. Supernatant in the centrifuge tubes wasused as heat treated crude LAL.

LAL for the measurement was prepared as described in Example 1 by usingthe heated crude LAL. A dilution series of endotoxin derived from E.coli O55:B5 between 1 ng/mL and 1000 ng/mL, and a dilution series ofCM-Pachyman between 1 ng/mL and 100 ng/mL were measured with LAL. Theprocedures of the measurement were same as described in Example 1.

Table 3 shows the reactivity of the heat treated LAL with endotoxin andβ-glucan by using the kinetic turbidimetric assay (KTA). β-glucan wasdetected with KTA, but endotoxin did not show any reactivity even at1000 ng/mL, again confirming the specificity of the assay for β-glucan.

TABLE 3 Reactivity of Heat Treated LAL With Endotoxin and β-glucan in aKinetic Turbidimetric Assay. Concentration Onset Time (sec) (ng/ml)Beta-Glucan Endotoxin 1 3698 >6000 10 1400 >6000 100 1425 >6000 1000 Notdone >6000

Example 7 β-Glucan Detection in Patients

Serum samples from patients with positive blood culture were diluted10-fold with water for injection, and the diluted serum samples wereincubated at 80° C. for 10 min. The pretreated samples were measuredwith lyophilized heat-treated LAL by the kinetic chromogenic method. Thelyophilized heat-treated LAL was prepared from the LAL described inExample 5. CM-pachyman was used as a β-glucan standard. Serum samplesfrom healthy subjects were also pretreated and measured as the samemanner.

As shown in Table 4, the β-glucan values of the serum samples from thepatients were higher than 100 pg/mL, and those from the healthy subjectswere less than 34 pg/mL. Since the results of blood culture of thepatients showed yeast (Candida) and fungus (Aspergillus), these patientswere proven as fungemia. Considering these results, the β-glucan assaywith the heat-treated LAL is useful for the diagnosis of deep mycosis.

TABLE 4 β-glucan Values in Serum Samples From Fungemia Patients SampleBlood culture β-glucan (pg/mL) Patient #1: Pulmonary disease Candidaalbicans 137 Patient #2: Pulmonary disease Aspergillus sp. 878 Patient#3 Candida albicans 275 Healthy subject #1 not available <34 Healthysubject #2 not available <34

Example 8 Decreases in Endotoxin and β-Glucan Reactivity as a Functionof Temperature and Heating Time

Analysis of the experiments described in Example 2 revealed that therates of inactivation of endotoxin and β-glucan reactivity as a functionof heating temperature have an exponential relationship that can bedepicted using an Arrhenius plot. Comparing the logarithm of the onsettimes versus the heating times shown in Tables 1 and 2 yielded theregression parameters shown in Tables 5 and 6. Using the slopes and thetemperatures, Arrhenius plots for endotoxin reactivity and β-glucanreactivity demonstrated a correlation between logarithm of the slopesand the temperature. As shown in FIG. 8, for each temperature tested,the natural log of the slope from Table 5 (“ln(Etx)”) or Table 6(“ln(BG)”) was plotted against 1000/Absolute temperature (in Kelvins),yielding correlations both for rates of inactivation of endotoxinreactivity and for rates of inactivation of β-glucan reactivity.

TABLE 5 Regression Parameters of Inactivation of Endotoxin Reactivity atSeveral Temperatures Incubation Log (onset time (sec)) time (h) 40° C.43° C. 45° C. 47° C. 50° C. Slope 0.040 0.708 1.019 4.120 12.595Y-intercept 2.524 2.390 2.613 2.223 2.390 Corr. Coefficient 0.999 1.0001.000 1.000 1.000 Temp 40 43 45 47 50

TABLE 6 Regression Parameters of Inactivation of β-glucan Reactivity atSeveral Temperatures Incubation time Log (onset time (sec)) (h) 40° C.43° C. 45° C. 47° C. 50° C. Slope −0.087 −0.175 0.094 0.217 3.599Y-intercept 2.483 2.536 2.471 2.554 2.471 Corr. Coefficient −0.971−1.000 0.998 0.608 0.937

Based on the observed relationships between rates of inactivation andheating times and temperatures, the heating time required to achieve acertain degree of inactivation was calculated for the lysate tested inExample 2. For example, a 1,000-fold reduction in the endotoxinreactivity of the lysate of Example 2 corresponds to a heating time of

t _(A1)=0.825*2.718^((56340/(T+273)))/(9.54*10⁷⁶)(hours).

A 1,000,000-fold reduction in endotoxin reactivity corresponds to aheating time twice as long:

t _(A2)=1.65*2.718^((56340/(T+273)))/(9.54*10⁷⁶)(hours).

Similarly, a 10-fold reduction in β-glucan in the lysate of Example 2 isobtained after a heating time of

t _(B1)=1.059*2.718^((76510/(T+273)))/(2.20*10¹⁰³)(hours).

Because endotoxin reactivity degrades more quickly that β-glucanreactivity upon exposure to moderate heating, this permits thepreparation of lysates with moderately or substantially reducedendotoxin reactivity. For example, Table 7 presents various proposedranges of heating times and temperatures permitting, for example, anendotoxin reactivity reduction of at least 1,000-fold and/or a β-glucanreactivity reduction of no more than about 10-fold (indicated inbold-face type).

TABLE 7 Estimated reactivity Heating time remaining Temperature (° C.)(minutes) Endotoxin β-glucan 40 750 0.108% 88.0% 40 13500 0.0000%  10.1%41 425 0.105% 85.4% 41 6200 0.000% 10.1% 42 240 0.106% 82.5% 42 29000.000% 9.7% 43 137 0.102% 78.9% 43 1350 0.000% 9.7% 44 78 0.103% 74.8%44 620 0.000% 10.0% 45 45 0.097% 70.0% 45 290 0.000% 10.0% 46 26 0.092%64.5% 46 135 0.000% 10.3% 47 15 0.091% 58.5% 47 64 0.000% 10.2% 48 8.50.104% 52.8% 48 31 0.000% 9.7% 49 5 0.094% 45.5% 49 15 0.000% 9.4% 502.9 0.096% 38.5% 50 7 0.000% 10.0% 51 1.7 0.094% 31.3% 51 3.4 0.000%9.8% 52 1 0.091% 24.4% 52 1.6 0.001% 10.4% 53 0.58 0.099% 18.5% 53 0.790.008% 10.1% 54 0.34 0.103% 13.2% 55 0.19 0.151% 9.9% 56 0.12 0.099%5.2% 57 0.07 0.114% 3.0% 58 0.042 0.110% 1.5% 59 0.025 0.115% 0.6% 600.015 0.116% 0.2%

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. The scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

INCORPORATION BY REFERENCE

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if the entire contents of each individual publication orpatent document was incorporated herein.

What is claimed is:
 1. A heat-treated Limulus amebocyte lysatecomprising (1→3)-β-D-glucan-sensitive Limulus Factor G and having anendotoxin reactivity less than 0.1% of the endotoxin reactivity of anuntreated Limulus amebocyte lysate.
 2. The Limulus amebocyte lysate ofclaim 1, wherein the Limulus amebocyte lysate is lyophilized.
 3. TheLimulus amebocyte lysate of claim 1, wherein the Limulus amebocytelysate retains sensitivity to 1 pg/mL carboxymethylated pachyman.
 4. TheLimulus amebocyte lysate of claim 3, wherein the Limulus amebocytelysate retains sensitivity to 0.1 pg/mL carboxymethylated pachyman.
 5. ALimulus amebocyte lysate according to claim 1, wherein the lysate isinsensitive to 200 EU/mL of Reference Standard Endotoxin.
 6. A Limulusamebocyte lysate according to claim 1, wherein the lysate is insensitiveto 0.01 mcg/mL LPS.
 7. The Limulus amebocyte lysate of claim 6, whereinthe lysate is insensitive to 0.1 mcg/mL LPS.
 8. The Limulus amebocytelysate of claim 7, wherein the lysate is insensitive to 1 mcg/mL LPS. 9.The Limulus amebocyte lysate of claim 8, wherein the lysate isinsensitive to 10 mcg/mL LPS.
 10. A composition comprising aheat-treated Limulus amebocyte lysate and a substrate.
 11. Thecomposition of claim 10, wherein the substrate is chromogenic.
 12. Thecomposition of claim 10, wherein the substrate is fluorogenic.
 13. Thecomposition of claim 10, wherein the composition is lyophilized.
 14. Amethod of detecting (1→3)-β-D-glucan in a sample, the method comprisingcombining the sample with a Limulus amebocyte lysate according to claim1 and detecting a change in an optical property of the sample.
 15. Themethod of claim 14, the method further comprising combining the samplewith a substrate prior to detecting the change in the optical propertyof the sample.